Efficient analysis of genes and gene expression will require sensitive, quantitative, high-throughput procedures that can simultaneously analyze many alleles with relatively low cost. A number of methods have been developed which permit the implementation of extremely sensitive diagnostic assays based on nucleic acid detection. Most of these methods employ exponential amplification of targets or probes. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Qβ replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics, 9:199-202 (1993)).
While all of these methods offer good sensitivity, with a practical limit of detection of about 100 target molecules, all of them suffer from relatively low precision in quantitative measurements. This lack of precision manifests itself most dramatically when the diagnostic assay is implemented in multiplex format, that is, in a format designed for the simultaneous detection of several different target sequences.
Fluorescence in situ hybridization is a useful method of determining the physical position of sequences relative to each other in the genome. However, the ability to detect sequences decreases as the size of the target sequence decreases so that detection of targets that are less than 500 bases in length is very difficult or impossible.
A number of different techniques have been developed specifically for the detection of single nucleotide polymorphisms (SNPs) or point mutations (PMs) in nucleic acids. These include solution-based assays using molecular beacon probes (Vet et al., Proc Natl Acad Sci USA 96(11):6394-9 (1999)); selective nuclease cleavage (Lyamichev et al., Nature Biotechnology 17, 292-296 (1999); Ryan et al., Mol. Diagn. 4:135-144 (1999)); direct DNA or cDNA sequence analysis (Wang et al., Science 280(5366):1077-82 (1998)); mass differential of allele discriminating oligonucleotides separated by time-of-flight mass spectrometry (Ross et al., Nat Biotechnol 16(13):1347-51 (1998); Tang et al., Proc Natl Acad Sci USA 96(18):10016-20 (1999)); differential electrophoretic mobility of DNA restriction fragments with zero or one mismatched base pair (Shi et al., Mol Diagn 4(4):343-51 (1999)); hybridization to oligonucleotide microarrays with allele discrimination being achieved by differential hybridization (Lipshutz et al., Bio Techniques. 19:442-447 (1995); Wang et al., Science 280(5366):1077-82 (1998)); selective DNA ligation (Shi et al., Mol Diagn 4(4):343-51 (1999)); and single nucleotide extension of the tethered oligonucleotide using mixture of four dideoxynucleotide triphosphates, each labeled with a distinct fluorophore (Picoult-Newberg et al., Genome Res 9(2):167-74 (1999)).
Each of these methods suffers from one or more drawbacks. First, both solution and electrophoretic mobility assays require prior synthesis of selected PCR amplicons or very large amounts of genomic DNA. They are also less amenable to high throughput formats. Direct sequence analysis of multiple loci in extensive pedigrees is both expensive and best conducted in dedicated sequencing laboratories. Mass spectrometry and oligonucleotide arrays offer considerable promise as high throughput SNP or PM analytical systems, but they too have technical limitations. The current detection sensitivities of array readers and Maldi-TOF spectrometers necessitate that each locus be amplified by PCR prior to analysis. Although oligonucleotide arrays can be constructed with 10×5 or more individual oligonucleotides per array, multiplexing PCR reactions beyond 100-200 primer pairs is extremely difficult and time consuming, thus the full analytical potential of large arrays is difficult to achieve. The current level of multiplexing of oligonucleotide mass tags is also in the 100-200 range. In the absence of better DNA amplification technology and/or a significant increase in detection sensitivity, both mass spectrometry and array analysis of SNPs and PMs will be sample size driven and dependent on prior DNA amplification.
Rolling Circle Amplification (RCA) driven by DNA polymerase can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (Lizardi et al., Nature Genet. 19: 225-232 (1998); U.S. Pat. No. 5,854,033 to Lizardi; PCT Application No. WO 97/19193). If a single primer is used, RCA generates in a few minutes a linear chain of hundreds or thousands of tandemly-linked DNA copies of a target that is covalently linked to that target. Generation of a linear amplification product permits both spatial resolution and accurate quantitation of a target. DNA generated by RCA can be labeled with fluorescent oligonucleotide tags that hybridize at multiple sites in the tandem DNA sequences. RCA can be used with fluorophore combinations designed for multiparametric color coding (PCT Application No. WO 97/19193), thereby markedly increasing the number of targets that can be analyzed simultaneously. RCA technologies can be used in solution, in situ and in microarrays. In solid phase formats, detection and quantitation can be achieved at the level of single molecules (Lizardi et al., 1998).
Ligation-mediated Rolling Circle Amplification (LM-RCA) involves circularization of a probe molecule hybridized to a target sequence and subsequent rolling circle amplification of the circular probe (U.S. Pat. No. 5,854,033 to Lizardi; PCT Application No. WO 97/19193). During amplification, the probe can become separated from the target sequence as it rolls. This can diminish the quality of spatial information obtained about the target.